glycosaminoglycan binding motif at s1/s2 proteolytic cleavage … · 1 glycosaminoglycan binding...

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Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site

on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2)

host cell entry

So Young Kim1,2*, Weihua Jin6, Amika Sood4, David W. Montgomery5, Oliver C. Grant5, Mark

M. Fuster1,2,3, Li Fu6, Jonathan S. Dordick7, Robert J. Woods5, Fuming Zhang7*, Robert J.

Linhardt6,7,8,9*

1 Department of Medicine, Division of Pulmonary and Critical Care, University of California San

Diego, La Jolla, California, United States of America

2 VA San Diego Healthcare System, Medical and Research Sections, La Jolla, California, United

States of America

3 Glycobiology Research and Training Center, University of California San Diego, La Jolla,

California, United States of America

4 Department of Biostatistics and Bioinformatics, Duke University, Durham, North Carolina,

United States of America

5 Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States

of America

6 Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New

York, United States of America

7 Department of Chemical and Biological Engineering, Center for Biotechnology and

Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States of

America

8 Department of Biological Science, Center for Biotechnology and Interdisciplinary Studies,

Rensselaer Polytechnic Institute, Troy, New York, United States of America

9 Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer

Polytechnic Institute, Troy, New York, United States of America

Corresponding Authors

*[email protected] (SYK), [email protected] (FZ), [email protected] (RJL)

was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 15, 2020. . https://doi.org/10.1101/2020.04.14.041459doi: bioRxiv preprint

mailto:[email protected]

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Abstract

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a

pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective

therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel

insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686

(PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein

(SGP) led us to hypothesize that host cell surface GAGs might be involved in host cell entry of

SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both

monomeric and trimeric SARS-CoV-2 spike more tightly bind to immobilized heparin (KD = 40

pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM,

respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant

heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP

binding to immobilized heparin were 0.056 μM, 0.12 μM, and 26.4 μM, respectively. Finally,

unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-

binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and

at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open

conformation. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances

carbohydrate-based COVID-19 therapeutic development.


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Introduction

In March 2020, the World Health Organization declared severe acute respiratory

syndrome-related coronavirus 2 (SARS-CoV-2) a pandemic less than three months after its initial

emergence in Wuhan, China [1,2]. SARS-CoV-2 is a zoonotic Betacoronavirus transmitted

through person-person contact through airborne and fecal-oral routes, and has caused over 693,000

confirmed coronavirus disease 2019 (COVID-19) cases and 33,000 associated deaths worldwide

[2–5]. While there is limited understanding of SARS-CoV-2 pathogenesis, extensive studies have

been performed on how its closely related cousins, SARS-CoV and MERS-CoV (Middle East

respiratory syndrome-related coronavirus), invade host cell. Upon initially contacting the surface

of a host cell, SARS-CoV and MERS-CoV exploit host cell proteases to prime their surface spike

glycoproteins (SGPs) for fusion activation, which is achieved by receptor binding, low pH, or both

[6,7]. The receptor binding domain (RBD) resides within subunit 1 (S1) while subunit 2 (S2)

facilitates viral-host cell membrane fusion [6]. Activated SGP undergoes a conformational change

followed by an initiated fusion reaction with the host cell membrane [6]. Endocytosed virions are

further processed by the endosomal protease cathepsin L in the late endosome [7,8]. Both MERS-

Cov and SARS-CoV require proteolytic cleavage at their S2’ site, but not at their S1-S2 junction,

for successful membrane fusion and host cell entry [6,7]. Additionally, receptors involved in fusion

activation of SARS-CoV and MERS-CoV include heparan sulfate (HS) and angiotensin-

converting enzyme 2 (ACE2), and dipeptidyl peptidase 4 (DPP4), respectively [9–11].

SARS-CoV and other pathogens arrive at a host cell surface by clinging, through their

surface proteins, to linear, sulfated polysaccharides called glycosaminoglycans (GAGs) [12–14].

The repeating disaccharide units of GAGs, comprised of a hexosamine and a uronic acid or a

galactose residue, are often sulfated (S1 Fig) [15]. GAGs are generally found covalently linked to


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core proteins as proteoglycans (PGs) and reside inside the cell, at the cell surface, and in the

extracellular matrix (ECM) [15]. GAGs facilitate various biological processes, including cellular

signaling, pathogenesis, and immunity, and possess diverse therapeutic applications [15]. For

example, an FDA approved anticoagulant heparin (HP) is a secretory GAG released from granules

of mast cells during infection [15,16]. Some GAG binding proteins can be identified by amino

acid sequences known as Cardin-Weintraub motifs corresponding to ‘XBBXBX’ and

‘XBBBXXBX’, where X is a hydropathic residue and B is a basic residue, such as arginine and

lysine, responsible for interacting with the sulfate groups present in GAGs [17,18]. Examination

of the SARS-CoV-2 SGP sequence revealed that the GAG-binding motif resides within S1-S2

proteolytic cleavage motif (furin cleavage motif BBXBB) that is not present in SARS-CoV or

MERS-CoV SGPs (Fig 1, S2 Fig, S3 Fig) [19]. Additionally, we discovered GAG-binding-like

motifs within RBD and S2’ proteolytic cleavage site in SARS-CoV-2 SGP (Fig 1, S2 Fig, S3 Fig).

This discovery prompted us to hypothesize that GAGs may contribute to SARS-CoV-2 fusion

activation and host cell entry as a novel mechanism through SGP binding. We performed surface

plasmon resonance (SPR)-based binding assays to determine binding kinetics of the interactions

between various GAGs and SARS-CoV-2 SGP in comparison with SARS-CoV, and MERS-CoV

SGP to address this question. Lastly, we performed blind docking on the trimeric SARS-CoV-2

SGP model to objectively identify the preferred binding GAG-binding sites on the SGP.


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Results

Kinetic measurements of CoV SPG-HP interactions

Previous reports showed that various CoV bind GAGs through their SGPs to invade host

cells [13]. In the current study, we utilized SPR to measure the binding kinetics and interaction

affinity of monomeric and trimeric SARS-CoV-2, monomeric SARS-CoV and MERS-CoV with

SGP-HP using a sensor chip with immobilized HP. Sensorgrams of CoV SGP-HP interactions are

shown in Fig 2. The sensorgrams were fit globally to obtain association rate constant (ka),

dissociation rate constant (kd) and equilibrium dissociation constant (KD) (Table 1) using the

BiaEvaluation software and assuming a 1:1 Langmuir model. SARS-CoV-2 and MERS CoV SGP

exhibited a markedly low dissociation rate constant (kd ~ 10-7 1/s) suggesting excellent binding

strength. The HP binding properties of monomeric SARS-CoV-2 SGP was comparable to that of

the trimeric form (KD of monomer and trimer were 40 pM and 73 pM, respectively). In comparison,

previously known HP binding SARS-CoV SGP showed nearly 10-fold lower affinity, 500 nM.

The extremely high binding affinity of SARS-CoV-2 SGP to HP was supported by the chip surface

regeneration conditions. The immobilized HP surface could only be regenerated using a harsh

regeneration reagent, 0.25% SDS, instead of the standard 2M NaCl solution used for removing

HP-binding proteins. One reason for SARS-CoV-2 SGP monomer and trimer extremely high

affinity to immobilized heparin is the high density of surface bound ligands might promote

polyvalent interactions. The difference of binding kinetics and affinity of CoV SGPs to HP may

also be due in part to the difference in protein sequence of the Cov SGPs. Based on amino acid

alignment analysis using the Basic Local Alignment Search Tool (BLAST), SARS-CoV and

SARS-CoV-2 SGPs share 76% similarity. Association rate constants (ka) for MERS-CoV SGP

(339 (± 27) 1/M-1s1) was the lowest, followed by monomeric and trimeric SARS-CoV-2 SGP (2.5


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× 103 (± 62.7) M-1s-1 and 1.6 × 103 (± 127) M-1s-1, respectively) (Table 1). SARS-CoV SGP had

the highest Ka, which was 4.12 × 104 (± 136) M-1s-1. The differences in ka values suggest a different

mechanism when each SGP binds HP in addition to differences in binding strengths.

Table 1 Summary of kinetic data of CoV SGP-HP interactions*

Interaction ka (M-1s1) kd (1/s) KD (M)

SARS-CoV-2 SGP

(monomer) 2.5 103

(±62.7)

1.0 10-7

(±7.9 10-8)

4.0 10-11

SARS-CoV-2 SGP

(trimer) 1.6 103

(±127)

1.2 10-7

(±5.5 10-8)

7.3 10-11

SARS-CoV SGP

(monomer) 4.12 104

(±136)

4.01 10-4

(±6.49 10-6)

5.0 10-7

MERS-CoV SGP

(monomer)

339

(±27) 3.5 10-7

(±2.6 10-6)

1.0 10-9

*The data with (±) in parentheses are the standard deviations (SD) from global fitting of five

injections.

SPR solution competition study on the interaction between surface-bound HP and SARS-

CoV-2 SGP to HP-derived oligosaccharides in solution

Solution/surface competition experiments were performed by SPR to examine the effect of

the saccharide chain length of HP on the SARS-CoV-2 SGP-HP interaction. HP-derived

oligosaccharides of different lengths, from tetrasaccharide (dp4) to octadecasaccharide (dp18),

were used in these competition studies. The same concentration (1000 nM) of HP oligosaccharides

were mixed in the SARS-CoV-2 SGP protein (50 nM)/ HP interaction solution. Negligible

competition was observed (S4 Fig) when 1000 nM of oligosaccharides (from dp4 to dp18) were


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present in the protein solution suggesting that the SARS-CoV-2 SGP-HP interaction is chain-

length dependent and it prefers to bind full chain (~dp30) HP.

SPR solution competition study of different chemically modified HP derivatives and GAGs

Competition levels measured by SPR for chemically modified HP derivatives are shown

in Fig 3. The results of these studies demonstrate that all the chemically modified HPs, N-

desulfated HP, 2-O-desulfated HP and 6-O-desulfated HP, were unable to compete with

immobilized HP for binding to SARS-CoV-2 SGP-HP suggesting all the sulfate groups within HP

have critical impact on this interaction.

SPR competition assay was also used to test the binding preference of SARS-CoV-2 SGP

for various GAGs (S1 Fig), including various chondroitin sulfates, dermatan and keratan sulfates,

and the results are shown in S5 Fig. Weak or no inhibitory activities were observed for all GAGs

tested, suggesting that the binding of SARS-CoV-2 SGP protein to GAGs appears to be HP

specific and greatly influenced by the level of sulfation within the GAG.

SPR solution competition dose response analysis of HP, Tri-sulfated HS and NACH

Solution competition dose response analysis between surface immobilized HP and various

soluble glycans (HP, non-anticoagulant trisulfated (TriS) HS, and non-anticoagulant low

molecular weight HP (NACH)) was performed to calculate their IC50 values (Figs 4A-4E). SARS-

CoV-2 SGP protein (50 nM) samples were pre-mixed with different concentrations of glycans

before injection into the HP chip. The sensorgrams (Figs 4A, 4C and 4E) show that once the active

binding sites on SARS-CoV-2 SGP were occupied by glycans in solution, the binding of SARS-

CoV-2 SGP to the surface-immobilized HP decreased resulting in a reduction of signal in a


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concentration dependent fashion. The IC50 values (concentration of competing analyte resulting

in a 50% decrease in RU) were calculated from the plots SARS-CoV-2 SGP binding signal

(normalized) vs. glycans concentration in solution (Figs 4B, 4D and 4F). The IC50 values of HP,

TriS HS and NACH were 0.056 µM, 012 µM and 26.4 µM, respectively.

Identification of GAG-binding motifs by blinding docking analysis

Using a modified version of Autodock Vina tuned for use with carbohydrates (Vina-Carb)

[20,21], we performed blind docking on the trimeric SARS-CoV-2 SGP model to discover

objectively the preferred binding GAG-binding sites on the SGP protein surface. The SGP

contains three putative GAG-binding motifs with the following sequences: 453-459 (YRLFRKS),

681-686 (PRRARS), and 810-816 (SKPSKRS), which we define as sites 1, 2, and 3, respectively

(Fig 1, S2 Fig, S3 Fig). An HS hexasaccharide fragment (GlcA(2S)-GlcNS(6S)) binds site 2 in

each monomer chain in the trimeric SGP (Fig 5C, S3 Fig). The docking results also indicates that

HS may bind to site 1 when the apex of the S1 monomer is in an open conformation, as this allows

basic residues to be more accessible to ligand binding. The site 1 residues are less accessible for

GAG binding when the domain is in a closed conformation (Fig 5D). The electrostatic potential

surface representation of the trimeric SGP confirms that the GAG-binding poses generally prefer

regions of positive charge, as expected, and illustrates that basic residues within site 3 are not

exposed for binding to HS on any of the chains (Fig 5A). Finally, our blind docking analysis

reveals that a longer HS polymer may span an inter-domain channel that contains site 2.


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Discussion

The original SARS-CoV and numerous pathogens exploit host cell surface GAGs during

the initial step of host cell entry [12–14]. Based on our discovery of GAG-binding and GAG-

binding-like motifs at site 1 (within the RBD, Y453-S459), site 2 (at the proteolytic cleavage site

at S1/S2 junction, P681-S686), and site 3 (at the S2’ proteolytic cleavage site, S810-S816), we

hypothesized that SARS-CoV-2 may also interact with host cell surface GAGs through its SGPs

to invade host cell (Fig 1, S2 Fig, S3 Fig). The predominant GAG in normal human lung is HS

followed by CS [22] and it is noteworthy that lung tissue is rich in mast cells and has been a source

of commercial HP [23]. Using unbiased docking, we found that TriS HS hexasaccharide

(GlcA(2S)-GlcNS(6S)) binds site 2 in each monomer chain in the trimeric SARS-CoV-2 SGP (Fig

5C). TriS HS hexasaccharide may additionally bind site 1 when the SGP monomer is in an open

conformation, but not site 3 where the basic residues are not accessible to the surface (Figs 5C and

5D). Docking results indicated that the HS hexasaccharides could span an inter-domain channel

that includes site 2, suggesting a mechanism for the binding of a longer HS sequence (Fig 5C).

Next, we experimentally determined binding kinetics for the interactions between HP (rich

(60-80%) in TriS domains) and monomeric SARS-CoV-2, trimeric SARS-CoV-2, monomeric

SARS-CoV, and monomeric MERS-CoV SGPs using SPR binding assays (Fig 2 and Table 1).

GAG-protein interactions are mainly electrostatically driven [24], thus, HS-binding proteins

generally bind HP due to its higher degree of sulfation [15]. We discovered that HP binds both

monomeric and trimeric SARS-CoV-2 SGP with remarkable affinity (KD = 40 pM and 73 pM,

respectively) (Fig 2 and Table 1). This was unexpectedly tight binding for a GAG-protein

interaction as even one of one of the prototypical HP-binding proteins, fibroblast growth factor 2

(FGF2), has a KD of 39 nM [25]. In comparison, SARS-Cov and MERS-CoV SGPs also bind HP,


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however, much more weakly with binding strengths of KD = 500 nM and 1 nM, respectively (Fig

2 and Table 1). While HS facilitates SARS-CoV host cell entry and is an essential host cell surface

receptor, its involvement in MERS-CoV host cell entry or binding kinetics for SARS-Cov and

MERS-CoV SGPs had not previously been reported [10].

After discovering the high binding affinity between HP and SARS-CoV-2 SGP, we next

found that the degree and position of sulfation within HP was important for its successful binding

to monomeric SARS-CoV-2 SGP (Figs 3 and 4). N-, 2-O, and 6-O-sulfation were all required for

binding to SARS-CoV-2 SGP (Fig 3). This was additionally demonstrated when competitive

binding studies gave IC50 values of HP (0.056 μM), TriS HS (NS2S6S) (0.12 μM), and NACH

(26.4 μM) for the inhibition of SARS-CoV-2 SGP binding to immobilized HP (Fig 4). Both HP

and TriS HS are sulfated at N-, 2-O-, and 6-O- positions and have approximately the same

molecular weight (and chain length) but HP has additional 3-O-sulfation, responsible for its

anticoagulant activity (S1 Fig). NACH lacks an intact antithrombin binding site, has a lower

average molecular weight of 5 kDa than HP, and a higher content (>90%) of TriS [26].

The low IC50 of these GAGs suggest that the FDA approved anticoagulant HP, or its non-

anticoagulant derivatives, might have therapeutic potential against SARS-CoV-2 infection as

competitive inhibitors. The location of proposed GAG-binding sites is also of interest. Unlike

SARS-CoV and MERS-CoV SGPs, SARS-CoV-2 SGP has a novel insert in the amino acid

sequence (681-686 (PRRARS)) that fully follows GAG-binding Cardin-Weintraub motif

(XBBXBX) and a furin-cleavage motif (BBXBB) at the S1/S2 junction (Fig 1). This site was also

shown to be a preferred GAG-binding motif by our unbiased docking study (Fig 5). Proteolytic

cleavage at S1/S2 is not required for successful viral-host cellular membrane fusion in SARS-CoV


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and MERS-CoV SGPs [6,7]. Proteolytic cleavage primes the SGP for fusion activation and may

additionally influence cell-cell fusion, host cell entry, and/or the infectivity of the virus [13,27].

Some CoVs, including mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV),

possess both GAG-binding and furin cleavage motifs at their S1/S2 junction in their SGPs [13]. In

the cases of MHV and IBV spike proteins, a single amino acid mutation near the GAG-binding

and furin cleavage motifs resulted from a cell culture adaptation, and determines whether a virion

binds GAGs or exploits host cell surface protease, but not both [28]. While not within the CoV

family, human immunodeficiency virus type 1 (HIV-1) requires HS-binding to achieve optimal

furin processing because HS binding allows selective exposure of furin cleavage site [29]. While

the idea of repurposing HP as COVID-19 therapeutic sounds appealing, further questions,

including in vitro relevance of GAGs as host cell surface receptors, proteolytic processing of SGPs

at S1/S2 junction, and their relationship in host cell entry and infectivity, must first be carefully

evaluated.

Based on our findings, we propose a model on how GAGs may facilitate host cell entry of

SARS-CoV-2 (Fig 6). First, virions land on the epithelial surface in the airway by binding to HS

through their SGPs (Fig 6A). Host cell surface proteoglycans utilize their long HS chains to

securely wrap around the trimeric SGP (Fig 6A). During this step, heavily sulfated HS chains span

inter-domain channel containing GAG-binding site 2 on each monomer in the trimeric SGP and

binds site 1 within the RBD in an open conformation (Fig 5). Host cell surface and extracellular

proteases, such as furin and transmembrane serine protease 2 (TMPRSS2), may process site 2

(S1/S2 junction) and/or 3 (S2’) and GAG chains come off from site 2 upon cleavage (Fig 6B). HS

and ACE2 binding to more readily accessible RBD containing site 1 may drive conformational

change of SGP and activate viral-cellular membrane fusion [30]. Finally, SGP on the endocytosed


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virion may utilize an endosomal host cell protease, such as cathepsin L, to further execute viral-

cellular membrane fusion. (Fig 6C).

In conclusion, we have discovered that GAGs can facilitate host cell entry of SARS-CoV-

2 by binding to SGP in the current work. SPR studies demonstrate that both monomeric and

trimeric SARS-CoV-2 SGP bind HP with remarkably high affinity and it prefers long, heavily

sulfated (TriS rich) structures. Additionally, we reported low IC50 of HP and derivatives against

HP and SARS-CoV-2 SGP interactions suggesting therapeutic potential of HP as COVID-19

competitive inhibitors. Lastly, unbiased computational ligand docking indicated that a TriS HS

oligosaccharide preferably interacts with GAG-binding motifs at the S1/S2 junction and within

receptor binding domain and hinted at mechanism of binding. This study adds to our current

understanding of SARS-CoV-2 pathogenesis and serves a foundation for designing glycoconjugate

vaccines and therapeutics to successfully contain and eliminate COVID-19.

Materials and methods

Materials

Monomeric SARS-CoV-2, SARS-CoV, and MERS-CoV SGPs were purchased from Sino

Biological Inc. Trimeric SARS-CoV-2 SGP was kindly provided by Prof. Jason McLellan

(University of Texas at Austin). The GAGs used in this study were porcine intestinal HP (HP)

(average molecular weight, Mw = 16 kDa) and porcine intestinal heparan sulfate (HS) (Mw = 14

kDa) from Celsus Laboratories (Cincinnati, OH); chondroitin sulfate A (CSA, Mw = 20 kDa) from

porcine rib cartilage (Sigma, St. Louis, MO), dermatan sulfate (DS, Mw = 30 kDa) from porcine

intestine (Sigma), chondroitin sulfate C (CSC, Mw = 20 kDa) from shark cartilage (Sigma),

chondroitin sulfate D (CSD, Mw = 20 kDa) from whale cartilage (Seikagaku, Tokyo, Japan) and


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chondroitin sulfate E (CSE, Mw = 20 kDa) from squid cartilage (Seikagaku). N-desulfated HP

(N-DeS HP, Mw = 14 kDa) and 2-O-desulfated HP (2-DeS HP, MW = 13 kDa) were prepared in

house based on protocols by Yates et al [31]. The 6-O-desulfated HP derivative, 6-DeS HP, Mw

= 13 kDa, was generously provided by Prof. Lianchun Wang (University of South Florida). Non-

anticoagulant low molecular weight HP (NACH) was synthesized from dalteparin, a nitrous acid

depolymerization product of porcine intestinal HP, followed by periodate oxidation as described

in our previous work [26]. TriS HS (NS2S6S) was synthesized from N-sulfo heparosan with

subsequent modification with C5-epimerase and 2-O- and 6-O-sulfotransferases (2OST and

6OST1/6OST3) [32]. HP oligosaccharides included tetrasaccharide (dp4), hexasaccharide (dp6),

octasaccharide (dp8), decasaccharide (dp10), dodecasaccharide (dp12), tetradecasaccharide

(dp14), hexadecasaccharide (dp16) and octadecasaccharide (dp18) and were prepared from

porcine intestinal HP controlled partial heparin lyase 1 treatment followed by size fractionation.

The chemical structures of the GAGs are shown in S1 Fig. Sensor SA chips were from GE

Healthcare (Uppsala, Sweden). SPR measurements were performed on a BIAcore 3000 operated

using BIAcore 3000 control and BIAevaluation software (version 4.0.1).

Preparation of HP biochip

Biotinylated HP was prepared by conjugating its reducing end to amine-PEG3-Biotin

(Pierce, Rockford, IL). In brief, HP (2 mg) and amine-PEG3-Biotin (2 mg, Pierce, Rockford, IL)

were dissolved in 200 µl H2O, 10 mg NaCNBH3 was added. The reaction mixture was heated at

70 °C for 24 h, after that a further 10 mg NaCNBH3 was added and the reaction was heated at

70 °C for another 24 h. After cooling to room temperature, the mixture was desalted with the spin

column (3,000 MWCO). Biotinylated HP was collected, freeze-dried and used for SA chip


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preparation. The biotinylated HP was immobilized to streptavidin (SA) chip based on the

manufacturer’s protocol. The successful immobilization of HP was confirmed by the observation

of a 200-resonance unit (RU) increase on the sensor chip. The control flow cell (FC1) was

prepared by 2 min injection with saturated biotin.

Measurement of interaction between HP and CoV SGP using BIAcore

SGP samples were diluted in buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005%

surfactant P20, pH 7.4,). Different dilutions of protein samples were injected at a flow rate of 30

µL/min. At the end of the sample injection, the same buffer was flowed over the sensor surface to

facilitate dissociation. After a 3 min dissociation time, the sensor surface was regenerated by

injecting with 30 µL of 0.25% sodium dodecyl sulfonate (SDS) to get fully regenerated surface.

The response was monitored as a function of time (sensorgram) at 25 °C.

Solution competition study between HP on chip surface and HP, HP-derived

oligosaccharides, chemically modified HP or GAGs in solution using SPR

SARS-CoV-2 SGP (50 nM) mixed with 1 M HP, HP-derived oligosaccharides,

chemically modified HP or GAGs in SPR buffer were injected over HP chip at a flow rate of 30

L/min, respectively. After each run, the dissociation and the regeneration were performed as

described above.

SPR solution competition IC50 measurement of glycans (HP, TriS HS and NACH) inhibition

on SARS-CoV-2 SGP-HP interaction


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Solution competition studies between surface HP and soluble glycans (HP, TriS HS and

NACH) to measure IC50 were performed using SPR [33]. In brief, SARS-CoV-2 S-protein (50

nM) samples alone or mixed with different concentrations of glycans in SPR buffer were injected

over the HP chip at a flow rate of 30 l/min, respectively. After each run, dissociation and

regeneration were performed as described above. For each set of competition experiments, a

control experiment (only protein without glycan) was performed to ensure the surface was

completely regenerated.

Protein modeling

The 3D coordinates for the SGP trimer (NCBI reference sequence YP_009724390.1) were

downloaded from the SWISS-MODEL homology modeling server [34]. The selected model was

generated with the Cryo-EM structure PDB ID 6VSB as a template, which has a 99.26% sequence

identity and 95% coverage for amino acids 27 to 1146. The template and resulting model is the

“prefusion” structure with one of the three receptor binding domains (Chain A) in the “up” or

“open” conformation [30]. Cryo-EM studies have revealed that the SARS-CoV-2 SGP

trimer exists in two conformational states in approximately equal abundance [35]. In one state, all

SGP monomers have their hACE2-binding domain closed, and in the other, one monomer has

its hACE2-binding domain open, where it is positioned away from the interior of the protein.

Ligand docking

Initial coordinates for a hexasaccharide fragment of HS (GlcA(2S)-GlcNS(6S))3 were

generated using the GAG-Builder tool [36] at GLYCAM-Web (glycam.org) and used for unbiased

(blind) docking. A hexasaccharide was chosen as being sufficiently long to represent a typical


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GAG length found in protein co-complexes [36] and to avoid introducing so many degrees of

internal flexibility that the efficiency of the docking conformational search algorithm was

impaired. Docking was performed using a version of Vina-Carb [21] that has been modified to

improve its performance for GAGs. A grid box with dimensions (x = 190, y = 223, z = 184 Å) was

placed at the geometric center the protein enclosing its entire surface. Docking was performed

with default values, with the following exceptions: exhaustiveness = 80, chi_cutoff = 2, and

chi_coeff = 0.5. All sulfate and hydroxyl groups and glycosidic torsion angles were treated as

flexible, resulting in 83 ligand poses.

Acknowledgements

We appreciate Prof. Jason McLellan from University of Texas Austin for providing trimeric

SARS-CoV-2 SGP. Additionally, we thank professor Lianchun Wang from University of South

Florida for providing 6-O-desulfated HP derivative.

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mission-briefing-on-covid-19---19-march-2020

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Figure legends

Fig 1. Identification of GAG-binding motif within SARS-CoV-2, SARS-CoV, and MERS-

CoV SGPs. Domains in SGP include signal peptide (SP), N-terminal domain (NTD), receptor-

binding domain (RBD), fusion peptide (FP), heptad repeat 1/2 (HR 1/2).

Fig 2. SPR sensorgrams for binding kinetics/affinity measurements for SGP-HP interactions.

(A) SARS-CoV-2 SGP (monomer), concentration of SGP (from top to bottom): 100, 50, 25, 12.5

and 6.25 nM. (B) SARS-CoV SGP, concentrations of SARS-CoV SGP (from top to bottom): 100,

50, 25, 12.5 and 6.25 nM. (C) MERS CoV SGP, concentrations of MERS CoV SGP (from top to

bottom): 100, 50, 25, 12.5 and 6.25 nM. (D) SARS-CoV-2 SGP (trimer), concentration of SGP

(from top to bottom): 800, 400, 200, 100 and 50 nM. The black curves are the fits using a 1:1

Langmuir model from BIAevaluate 4.0.1.

Fig 3. Bar graphs of normalized SARS-CoV-2 SGP binding preference to surface HP by

competing with different chemical modified HP in solution. Concentration was 50 nM for

SARS-CoV-2 SGP and 1000 nM for different chemical modified HP. All bar graphs based on

triplicate experiments.

Fig 4. Inhibition analysis of glycans on the interactions between SARS-CoV-2 SGP and HP

using SPR; SARS-CoV-2 SGP concentration was 50 nM. (A) Competition SPR sensorgrams of

SARS-CoV-2 SGP-HP interaction inhibiting by different concentration of heparin. (B) Dose

response curves for IC50 calculation of heparin using SARS-CoV-2 SGP inhibition data from

surface competition SPR. (C) Competition SPR sensorgrams of SARS-CoV-2 SGP-HP interaction


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inhibiting by different concentration of TriS HS. (D) Dose response curves for IC50 calculation of

TriS HS using SARS-CoV-2 SGP inhibition data from surface competition SPR. (E) Competition

SPR sensorgrams of SARS-CoV-2 SGP-HP interaction inhibiting by different concentration of

NACH. (F) Dose response curves for IC50 calculation of NACH using SARS-CoV-2 SGP

inhibition data from surface competition SPR.

Fig 5. Structure of trimeric SARS-CoV-2 SGP and proposed GAG-binding motifs. (A)

Electrostatic potential surface (-ve charge (red) to +ve charge (blue)) computed with Chimera. (B)

Electrostatic potential surface showing a top view of the SGP trimer. (C) Solvent accessible surface

of the SARS-CoV-2 SGP trimer (pink (Chain A), grey (Chain B), blue (Chain C)) showing the

predicted poses of HS hexasaccharides (orange) obtained from unbiased docking, and the three

GAG-binding motifs (yellow (Chain A), white (Chain B), red (Chain C)), image generated with

VMD [37]. (D) Solvent accessible surface showing a top view of the SGP trimer. Amino acid

sequences for GAG-binding motifs site 1, 2, and 3 are YRLFRKS, PRRARS, and SKPSKRS.

Fig 6. Proposed model of SARS-CoV-2 host cell entry. SARS-CoV-2 surface is decorated with

envelop (E), membrane (M), and SGP [38]. (A) Virion lands on host cell surface by binding to

heparan sulfate proteoglycan (HSPG). (B) SGP goes through proteolytic digestion by host cell

surface protease, which initiates viral-host cell membrane fusion by conformational change caused

by host cell receptor binding (HSPG and ACE2). (C) Virion enters the host cell and may further

experience proteolytic processing by endosomal host cell protease.


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Fig 1.


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Fig 2.

-100

0

100

200

300

400

500

600

-50 0 50 100 150 200 250 300 350 400

RU

Re

sp

. D

iff.

sTim e

A

D

B

C


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Fig 3.

0

20

40

60

80

100

120

140

Control Hep N-Des Hep 2-Des Hep 6-Des Hep

No

rma

lize

d S

AR

S-C

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GP

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din

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Fig 4.


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Fig 5.

A

C

B

D

Site 3

Site 2

Site 1

Site 1


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Fig 6.


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glycosaminoglycan binding motif at s1/s2 proteolytic cleavage … · 1 glycosaminoglycan binding...

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